Linaclotide compositions

ABSTRACT

The present invention is directed to stable linaclotide compositions and methods of treating gastrointestinal disorders in patients in need thereof by providing the stable linaclotide compositions.

FIELD OF THE INVENTION

This invention relates to linaclotide compositions and methods fortreating gastrointestinal disorders.

BACKGROUND

U.S. Pat. Nos. 7,304,036 and 7,371,727 disclose peptides that act asagonists of the guanylate cyclase C (GC-C) receptor for the treatment ofgastrointestinal disorders. One particular peptide disclosed islinaclotide, which consists of the following amino acid sequence (SEQ IDNO: 1):

The '036 and '727 patents also disclose methods for preparinglinaclotide and related peptides. The contents of these patents areincorporated herein by reference in their entirety.

There remains a need for improved linaclotide compositions that haveimproved stability against formaldehyde, which can enter linaclotidecompositions from a variety of sources.

The present invention seeks to provide such improved linaclotidecompositions, as well as methods of treating gastrointestinal disordersby providing the linaclotide compositions to patients in need thereof.

SUMMARY OF THE INVENTION

The present invention relates in some embodiments to stable linaclotidecompositions that comprises linaclotide, a sterically hindered primaryamine, a divalent metal cation and a formaldehyde scavenger compound.

In some embodiments, the present invention relates to stable linaclotidecompositions that comprises linaclotide, a sterically hindered primaryamine, a divalent metal cation and a formaldehyde scavenger compound,and a peptide having the structure of formula (I) or a pharmaceuticallyacceptable salt thereof (formula I disclosed as SEQ ID NO: 3):

In some embodiments, the present invention relates to stable linaclotidecompositions that comprises linaclotide, a sterically hindered primaryamine, a divalent metal cation and a formaldehyde scavenger compound,and a peptide having the structure of formula (I) or a pharmaceuticallyacceptable salt thereof (formula VI disclosed as SEQ ID NO: 2):

In some embodiments, the present invention relates to stable linaclotidecompositions that comprises linaclotide, a sterically hindered primaryamine, a divalent metal cation and a formaldehyde scavenger compound,and a peptide having the structure of formula (IV) or a pharmaceuticallyacceptable salt thereof (formula VII disclosed as SEQ ID NO: 18):

In some embodiments, the present invention relates to stable linaclotidecompositions that comprises linaclotide, a sterically hindered primaryamine, a divalent metal cation and a formaldehyde scavenger compound,and one or both of the peptide of formula (VI) and the peptide offormula (VII).

In some embodiments, the present invention relates to stable linaclotidecompositions that comprises linaclotide, a sterically hindered primaryamine, a divalent metal cation, a formaldehyde scavenger compound, apeptide having the structure of formula (I) or a pharmaceuticallyacceptable salt thereof, and one or both of the peptide of formula (VI)and the peptide of formula (VII).

In some embodiments, the present invention relates to stable linaclotidecompositions that comprises linaclotide, a sterically hindered primaryamine, a divalent metal cation and a peptide having the followingstructure of formula (VIII) or a pharmaceutically acceptable saltthereof (formula VIII disclosed as SEQ ID NO: 3):

In some embodiments, the present invention relates to stable linaclotidecompositions that comprises linaclotide, a sterically hindered primaryamine, a divalent metal cation, a formaldehyde scavenger compound, apeptide having the structure of formula (I) and one or both of thepeptide of formula (III) and the peptide of formula (IV).

In some embodiments, the present invention relates to stable linaclotidecompositions that comprise linaclotide, a sterically hindered primaryamine, a divalent metal cation and a formaldehyde scavenger compound, apeptide having the structure of formula (I) or a pharmaceuticallyacceptable salt thereof (formula I disclosed as SEQ ID NO: 3):

and a peptide having a structure of Formula (II) or a pharmaceuticallyacceptable salt thereof (formula II disclosed as SEQ ID NO: 4):

In some embodiments, the present invention relates to stablecompositions that comprise linaclotide or a pharmaceutically acceptablesalt thereof, a peptide or a pharmaceutically acceptable salt thereofthat comprises the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn ProAla Cys Thr Gly Cys Tyr (SEQ ID NO: 25) (wherein at least one carboxylgroup of the peptide is an alkyl ester having the formula (-COOR) inwhich R is a C₁₋₆ alkyl), and one or more formaldehyde scavengercompounds.

In some embodiments, a stable linaclotide composition is provided whichcomprises linaclotide or a pharmaceutically acceptable salt thereof, apeptide having the structure of formula (I) or a pharmaceuticallyacceptable salt thereof, and one or more formaldehyde scavengercompounds (formula I disclosed as SEQ ID NO: 3):

In some embodiments, a stable linaclotide composition is provided whichcomprises linaclotide or a pharmaceutically acceptable salt thereof, apeptide (e.g., a formaldehyde imine product, e.g., a formaldehyde imineproduct) having the structure of formula (II) or a pharmaceuticallyacceptable salt thereof, and one or more formaldehyde scavengercompounds (formula II disclosed as SEQ ID NO: 8):

In some embodiments, a stable linaclotide composition is provided whichcomprises linaclotide, one or more formaldehyde scavenger compound, afirst peptide having the structure of formula (I) or a pharmaceuticallyacceptable salt thereof (formula I disclosed as SEQ ID NO: 3):

and a second peptide having the structure of Formula (II) or apharmaceutically acceptable salt thereof (formula II disclosed as SEQ IDNO: 4):

In some embodiments, a stable linaclotide composition is provided whichcomprises linaclotide, one or more formaldehyde scavenger compound, afirst peptide having the structure of formula (I) or a pharmaceuticallyacceptable salt thereof, and one or both of (i) a peptide having thestructure of formula (III) and (ii) a peptide having the structure offormula (IV).

In some embodiments, the linaclotide composition comprises a linaclotidepeptide comprising the amino acid sequence Cys Cys Glu Tyr Cys Cys AsnPro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 3) which is modified with theaddition of methylene at the α-amine group of the N-terminal Cys₁ whichis cross-linked to the amine group of Cyst to form an imidazolidinone 5membered ring at the N-terminus of the peptide (“Cys₁-IMD”), wherein“Cys₁-IMD” refers to the linaclotide imidazolidinone derivative modifiedon its N-terminal amine group. In some embodiments, the iminemodification may be produced by a formaldehyde mediated reaction in thepresence of acid catalyst.

In some embodiments, stable compositions are provided which compriselinaclotide or a pharmaceutically acceptable salt thereof, a peptidehaving the structure of formula (III) or a pharmaceutically acceptablesalt thereof, and one or more formaldehyde scavenger compounds (formulaIII disclosed as SEQ ID NO: 19):

wherein R′ is H or a C₁₋₆ alkyl, and at least one R′ is C₁₋₆ alkyl.

In some embodiments, stable compositions are provided which compriselinaclotide or a pharmaceutically acceptable salt thereof, a peptidehaving the structure of formula (IV) or a pharmaceutically acceptablesalt thereof, and one or more formaldehyde scavenger compounds (formulaIV disclosed as SEQ ID NO: 6):

wherein R is a C₁₋₆ alkyl.

In some embodiments, stable compositions are provided which compriselinaclotide or a pharmaceutically acceptable salt thereof, a peptidehaving the structure of formula (V) or a pharmaceutically acceptablesalt thereof, and one or more formaldehyde scavenger compounds (formulaV disclosed as SEQ ID NO: 7):

wherein R is C₁₋₆ alkyl.

Another aspect of the present invention provides a method for treating agastrointestinal disorder, which includes providing the linaclotidecomposition to a patient diagnosed with a gastrointestinal disorder.

The details of one or more embodiments of the invention are set forth inthe accompanying description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the dose response of exemplary peptides of the presentinvention in a T84 cell c-GMP assay.

FIG. 2 demonstrates an example of an analysis of exemplary peptides byRP-HPLC, wherein “Cyst-IMD” refers to the linaclotide imidazolidinonederivative modified on its N-terminal amine group.

FIG. 3 shows specific binding of linaclotide and Cys₁-IMD tocell-surface GC-C receptors on T84 cells in a competitive radioligandbinding assay.

FIG. 4 shows the dose response of exemplary peptides of the presentinvention in a T84 cell c-GMP assay.

The figures are provided by way of example and are not intended to limitthe scope of the present invention.

DETAILED DESCRIPTION

Guanylate cyclase C (GC-C) is a transmembrane receptor that is locatedon the apical surface of epithelial cells in the stomach and intestine.The receptor has an extracellular ligand-binding domain, a singletransmembrane region and a C-terminal guanylyl cyclase domain. When aligand binds to the extracellular domain of GC-C, the intracellularcatalytic domain catalyzes the production of cGMP from GTP. In vivo,this increase in intracellular cGMP initiates a cascade of events thatleads to increased secretion of chloride and bicarbonate into theintestinal lumen, increased luminal pH, decreased luminal sodiumabsorption, increased fluid secretion, and acceleration of intestinaltransit. cGMP is secreted bidirectionally from the epithelium into themucosa and lumen. The peptides and compositions of the present inventionbind to the intestinal GC-C receptor which is a regulator of fluid andelectrolyte balance in the intestine.

Formaldehyde Scavenger Compounds

It has been discovered that the stability of linaclotide compositionscan be increased to a surprisingly high degree by including in thelinaclotide composition a suitable/appropriate amount of one or moreformaldehyde scavenger compounds.

While not wishing to be bound by any theory, it is believed that theformaldehyde scavenger compounds increase linaclotide stability byreacting with formaldehyde in such a manner that the formaldehyde has alesser capacity/ability to react with linaclotide. In this regard, theformaldehyde scavenger compound can be any compound that reducesexposure of the linaclotide to formaldehyde in the composition, such asby reacting or interacting with at least a portion of formaldehyde thatenters the linaclotide composition or gets proximate to linaclotide.Such formaldehyde can enter the linaclotide composition from a varietyof sources and otherwise have deleterious effects on linaclotide anddrug product stability.

In some preferred embodiments, the formaldehyde scavenger compoundcomprises a nitrogen center that is reactive with formaldehyde, such asto form one or more reversible or irreversible bonds between theformaldehyde scavenger compound and the formaldehyde (preferably in someembodiments one or more irreversible covalent bonds). For example, insome preferred embodiments, the formaldehyde scavenger compoundcomprises one or more nitrogen atoms/centers that are reactive withformaldehyde to form a schiff base imine that is capable of subsequentlybinding with formaldehyde. In some preferred embodiments, theformaldehyde scavenger compound(s) comprise one or more nitrogen centersthat are reactive with formaldehyde to form one or more 5-8 membercyclic rings. In this regard, the formaldehyde scavenger compoundpreferably comprises one or more amine or amide groups. For example, theformaldehyde scavenger compound can be an amino acid, an amino sugar, analpha amine compound, or a conjugate or derivative thereof, or a mixturethereof. In some preferred embodiments, the formaldehyde scavengercompound comprises two or more amines and/or amides.

It has also been surprisingly discovered in some embodiments, thatformaldehyde scavenger compounds that comprise multiple amine bindingsites achieve surprisingly high stabilizing effects on linaclotide as isdemonstrated in Example 5. For example, some preferred formaldehydescavenger compounds comprise one or more (e.g., two or more) primaryamines. These include, for example, glycine, alanine, serine, threonine,cysteine, valine, lecuine, isoleucine, methionine, phenylalanine,tyrosine, aspartic acid, glutamic acid, arginine, lysine, ornithine,citrulline, taurine pyrrolysine, or a conjugate or mixture thereof.

In some especially preferred embodiments, the formaldehyde scavengercompound(s) comprises one or more secondary amines (which have beenfound in some embodiments to have higher reactivity with formaldehydeand thus higher stabilizing effects on linaclotide drug products). Someespecially preferred formaldehyde scavenger compounds comprise two ormore secondary amines. These include, for example, megulmine, histidine,aspartame, proline, tryptophan, citrulline, pyrrolysine, or a conjugateor mixture thereof

Other preferred formaldehyde scavenger compounds comprise one or more(e.g., two or more) primary amines and one or more (e.g., two or more)secondary amines. These include, for example, arginine, citrulline,pyrrolysine, or a conjugate or mixture thereof.

Moreover, the formaldehyde scavenger compounds can comprise one or moreamide groups. For example, the scavenger compound can be asparagine,glutamine, citrulline, or a conjugate or mixture thereof.

The formaldehyde scavenger compound can be introduced into thelinaclotide composition in any suitable form. For example, the scavengercan be lyophilized. Alternatively, or in addition, the formaldehydescavenger compound can be applied (e.g., sprayed) onto beads beforebeing introduced into the linaclotide composition. These beads can bemade of any suitable material, such as for example cellulose, glass,sugar(s) or a combination or mixture thereof.

In some embodiments, it has been surprisingly found (see Example 5) thatformaldehyde scavenger compounds may have higher stabilizing effects onlinaclotide when the formaldehyde scavenger compound used is inamorphous form. Therefore, in some especially preferred embodiments, theformaldehyde scavenger compound is amorphous.

Lastly, the formaldehyde scavenger compound can be present in thelinaclotide composition in any suitable amount. In some preferredembodiments, the linaclotide composition comprises a ratio of scavengerto leucine between about 10:1 and about 1:1, preferably between about7:1 and about 1:1, even more preferably between about 5:1 and about 1:1or even between about 3:1 and about 1:1.

Exemplary Peptides in the Linaclotide Composition:

The linaclotide compositions may further comprise one or more of thepeptides described herein.

In various embodiments, the linaclotide composition comprises a peptidethat is modified wherein at least one carboxyl group of the amino acidresidue of the peptide is modified to an alkyl ester. This modificationmay be produced, for example, by treating a carboxylic acid with analcohol in the presence of a dehydrating agent wherein the dehydratingagent can include but is not limited to a strong acid such as sulfuricacid. Other methods of producing alkyl esters from carboxyl groups arereadily known in those skilled in the arts and are incorporated herein.

As used herein, a carboxyl group has the formula: (—COOH).

As used herein, the term “alkyl”, refers to a saturated linear orbranched-chain monovalent hydrocarbon radical.

As used herein, a group is terminal or terminus when the group ispresent at the end of the amino acid sequence.

As used herein, an amine group on a peptide has the formula:

wherein R² is the rest of the peptide.

As used herein, an imine group on a peptide has the formula:

wherein R² is the rest of the peptide.

In some embodiments, the carboxylic acid of the side chain of aglutamate amino acid in a peptide sequence is modified into an alkylester.

In further embodiments, the carboxylic acid on the side chain of aglutamate amino acid a peptide sequence is modified into an ethyl ester.

In other embodiments, the C-terminus carboxylic acid of a tyrosine aminoacid in a peptide sequence is modified into an alkyl ester.

In further embodiments, the C-terminus carboxylic acid of a tyrosineamino acid of a peptide sequence is modified into an ethyl ester.

In some embodiments, the linaclotide composition comprises a peptide ora pharmaceutically acceptable salt thereof, wherein the peptidecomprises the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro AlaCys Thr Gly Cys Tyr (SEQ ID NO: 25), wherein at least one carboxyl groupof the peptide is an alkyl ester having the formula (—COOR) in which Ris a C₁₋₆ alkyl.

In several embodiments, the linaclotide composition comprises a peptidehaving an amino acid structure of (SEQ ID NO: 19):

or a pharmaceutically acceptable salt thereof, wherein R′ is H or a C₁₋₆alkyl, and at least one R′ is C₁₋₆ alkyl.

In some embodiments, the linaclotide composition comprises a peptidehaving an amino acid structure of (SEQ ID NO: 6):

wherein R is a C₁₋₆ alkyl (“Glu3-alkyl ester”).

In other embodiments, R is a C₁₋₄ alkyl.

In further embodiments, R is methyl, ethyl, or propyl.

In some embodiments, the linaclotide composition comprises a peptidehaving an amino acid structure of (“Glu3-ethyl ester”) (SEQ ID NO: 10):

In some embodiments, the linaclotide composition comprises a peptidehaving an amino acid structure of (SEQ ID NO: 11):

In some embodiments, the linaclotide composition comprises a peptidehaving an amino acid structure of (SEQ ID NO: 9):

In some embodiments, the C-terminal tyrosine of the Glu3-alkyl ester orpharmaceutically acceptable salt is absent.

In some embodiments, the linaclotide composition comprises a peptidehaving an amino acid structure of (SEQ ID NO: 7):

wherein R is C₁₋₆ alkyl.

In other embodiments, R is a C₁₋₄ alkyl.

In further embodiments, R is methyl, ethyl, or propyl.

In some embodiments, the linaclotide composition comprises a peptidehaving an amino acid structure of (“Tyr₁₄-ethyl ester”) (SEQ ID NO: 13):

In some embodiments, the linaclotide composition comprises a peptidehaving an amino acid structure of (SEQ ID NO: 14):

In some embodiments, the linaclotide composition comprises a peptidehaving an amino acid structure of (SEQ ID NO: 12):

In addition, the linaclotide composition may comprise a peptide that ismodified wherein at least one amine group of the amino acid residues ofthe peptide is modified into an imine. This modification may beproduced, for example, by treating an amine group with a carbonyl, suchas an aldehyde or ketone, in the presence of acid catalyst. Othermethods of producing imines from amine groups are readily known to thoseskilled in the arts and are incorporated herein.

In some embodiments, the imine modification may be produced by aformaldehyde mediated reaction in the presence of acid catalyst.

In further embodiments, the linaclotide composition comprises a peptidehaving an imine carbon that is cross-linked to another amine group ofthe peptide.

In other embodiments, the linaclotide composition comprises a peptidethat is modified into an imine at the α-amine group of the N-terminalamino acid, wherein the imine carbon is cross-linked with an amine groupof the second amino acid residue of the peptide forming a five memberedring.

In other embodiments, the linaclotide composition comprises a peptidehaving the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala CysThr Gly Cys Tyr (SEQ ID NO: 3) wherein the peptide may be modified withthe addition of methylene at the α-amine group of the N-terminal Cys₁which is cross-linked to the amine group of Cys₂ to form animidazolidinone 5 membered ring at the N-terminus of the peptide(“Cys₁-IMD”).

In several embodiments, the linaclotide composition comprises a peptideor a pharmaceutically acceptable salt thereof, wherein the peptidecomprising the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro AlaCys Thr Gly Cys Tyr (SEQ ID NO: 8), wherein at least one amine group ofthe peptide is an imine having the formula

wherein R² is the rest of the peptide.

In some embodiments, the linaclotide composition comprises a peptide ora pharmaceutically acceptable salt comprises a peptide wherein theN-terminal amine group of the peptide is an imine having the formula

wherein R² is the rest of the peptide.

In further embodiments, the linaclotide composition comprises a peptideor pharmaceutically acceptable salt thereof comprising an amino acidstructure of (SEQ ID NO: 8):

In several embodiments, the linaclotide composition comprises a peptideor pharmaceutically acceptable salt thereof comprising an amino acidstructure of (SEQ ID NO: 3):

In some embodiments, the C-terminal tyrosine of the Cys₁-IMD peptide orpharmaceutically acceptable salt thereof is absent. In some embodiments,the Cys₁-IMD peptide or pharmaceutically acceptable salt thereof furthercomprises one or more peptide modifications, wherein at least onecarboxyl group of the peptide is an alkyl ester having the formula(—COOR) in which R is a C₁₋₆ alkyl.

In several embodiments, the linaclotide composition comprises a peptideor a pharmaceutically acceptable salt thereof, wherein the peptideconsists of the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro AlaCys Thr Gly Cys Tyr (SEQ ID NO: 25), wherein at least one carboxyl groupof the peptide is an alkyl ester having the formula (—COOR) in which Ris a C₁₋₆ alkyl.

In some embodiments, the linaclotide composition comprises linaclotide,a sterically hindered primary amine, a divalent metal cation and aformaldehyde scavenger compound, and a peptide having the structure offormula (I) or a pharmaceutically acceptable salt thereof (formula VIdisclosed as SEQ ID NO: 2):

In some embodiments, the linaclotide composition comprises linaclotide,a sterically hindered primary amine, a divalent metal cation and aformaldehyde scavenger compound, and a peptide having the structure offormula (IV) or a pharmaceutically acceptable salt thereof (formula VIIdisclosed as SEQ ID NO: 18):

In some embodiments, the linaclotide composition comprises linaclotide,a sterically hindered primary amine, a divalent metal cation and aformaldehyde scavenger compound, and one or both of the peptide offormula (VI) and the peptide of formula (VII).

In some embodiments, the linaclotide composition comprises linaclotide,a sterically hindered primary amine, a divalent metal cation, aformaldehyde scavenger compound, a peptide having the structure offormula (I) or a pharmaceutically acceptable salt thereof, and one orboth of the peptide of formula (VI) and the peptide of formula (VII).

In some embodiments, the linaclotide composition comprises linaclotide,a sterically hindered primary amine, a divalent metal cation, aformaldehyde scavenger compound, a peptide having the followingstructure of formula (VIII) or a pharmaceutically acceptable saltthereof (formula VIII disclosed as SEQ ID NO: 3):

In several embodiments, the linaclotide composition comprises a peptidethat consists of an amino acid structure of (SEQ ID NO: 19):

or a pharmaceutically acceptable salt thereof, wherein R′ is H or a C₁₋₆alkyl, and at least one R′ is C₁₋₆ alkyl.

In some embodiments, the linaclotide composition comprises a peptide orpharmaceutically acceptable salt thereof that consists of an amino acidstructure of (SEQ ID NO: 6):

wherein R is a C₁₋₆ alkyl. In further embodiments, the C-terminaltyrosine is absent.

In other embodiments, R is a C₁₋₄ alkyl.

In further embodiments, R is methyl, ethyl, or propyl.

In some embodiments, the linaclotide composition comprises a peptide orpharmaceutically acceptable salt thereof that consists of a peptidehaving an amino acid structure of (SEQ ID NO: 10):

In some embodiments, the linaclotide composition comprises a peptide orpharmaceutically acceptable salt thereof that consists of a peptidehaving an amino acid structure of (SEQ ID NO: 11):

In some embodiments, the linaclotide composition comprises a peptide orpharmaceutically acceptable salt thereof that consists of a peptidehaving an amino acid structure of (SEQ ID NO: 9):

In some embodiments, the linaclotide composition comprises a peptide orpharmaceutically acceptable salt thereof wherein the peptide has anamino acid structure of (SEQ ID NO: 7):

wherein R is C₁₋₆ alkyl.

In other embodiments, R is a C₁₋₄ alkyl.

In further embodiments, R is methyl, ethyl, or propyl.

In some embodiments, the linaclotide composition comprises a peptide orpharmaceutically acceptable salt thereof that consists of an amino acidstructure of (SEQ ID NO: 13):

In some embodiments, the linaclotide composition comprises a peptide orpharmaceutically acceptable salt thereof that consists of an amino acidstructure of (SEQ ID NO: 14):

In some embodiments, the linaclotide composition comprises a peptide orpharmaceutically acceptable salt thereof that consists of an amino acidstructure of (SEQ ID NO: 12):

In several embodiments, the linaclotide composition comprises a peptideor a pharmaceutically acceptable salt thereof, wherein the peptideconsists of the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro AlaCys Thr Gly Cys Tyr (SEQ ID NO: 8), wherein at least one amine group ofthe peptide is an imine having the formula

wherein R² is the rest of the peptide.

In some embodiments, the linaclotide composition comprises a peptide ora pharmaceutically acceptable salt thereof having the structure offormula (III) (SEQ ID NO: 2):

In some embodiments, the linaclotide composition comprises a peptide ora pharmaceutically acceptable salt thereof having the structure offormula (IV) (SEQ ID NO: 18):

In some embodiments, the linaclotide composition comprises one or bothof a peptide or a pharmaceutically acceptable salt thereof having thestructure of formula (III) and a peptide having the structure of formula(IV).

In some embodiments, the linaclotide composition comprises a peptide ora pharmaceutically acceptable salt thereof having the structure offormula (III) and a peptide or a pharmaceutically acceptable saltthereof having the structure of formula (IV).

In some embodiments, the linaclotide composition comprises a peptide ora pharmaceutically acceptable salt that consists of a peptide whereinthe N-terminal amine group of the peptide is an imine having the formula

wherein R² is the rest of the peptide.

In further embodiments, the linaclotide composition comprises a peptideor pharmaceutically acceptable salt thereof that consists of an aminoacid structure of (SEQ ID NO: 8):

In some embodiments, the linaclotide composition comprises a peptide orpharmaceutically acceptable salt thereof that consists of an amino acidstructure of (SEQ ID NO: 3):

In some embodiments, the linaclotide composition comprises the Cys₁-IMDpeptide or pharmaceutically acceptable salt wherein the C-terminaltyrosine of the Cys₁-IMD peptide or pharmaceutically acceptable saltthereof is absent. In some embodiments, the Cys₁IMD peptide orpharmaceutically acceptable salt thereof further comprises one or morepeptide modifications, e.g., wherein at least one carboxyl group of thepeptide is an alkyl ester having the formula (—COOR), wherein R is aC₁₋₆ alkyl.

Production of Peptides

The peptides or precursor peptides described herein can be produced inany suitable manner such as recombinantly in any known proteinexpression system, including, without limitation, bacteria (e.g., E.coli or Bacillus subtilis), insect cell systems (e.g., Drosophila Sf9cell systems), yeast cell systems (e.g., S. cerevisiae, S.saccharomyces) or filamentous fungal expression systems, or animal cellexpression systems (e.g., mammalian cell expression systems). Peptidesor precursor peptides of the invention may also be chemicallysynthesized.

If the peptide or variant peptide is to be produced recombinantly, e.g.,E. coli, the nucleic acid molecule encoding the peptide may also encodea leader sequence that permits the secretion of the mature peptide fromthe cell. Thus, the sequence encoding the peptide can include the presequence and the pro sequence of, for example, a naturally-occurringbacterial ST peptide. The secreted, mature peptide can be purified fromthe culture medium.

The sequence encoding a peptide described herein can be inserted into avector capable of delivering and maintaining the nucleic acid moleculein a bacterial cell. The DNA molecule may be inserted into anautonomously replicating vector (suitable vectors include, for example,pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleic acid maybe a bacterial or bacteriophage DNA such as bacteriophage lambda or M13and derivatives thereof. Construction of a vector containing a nucleicacid described herein can be followed by transformation of a host cellsuch as a bacterium. Suitable bacterial hosts include but are notlimited to, E. coli, B. subtilis, Pseudomonas and Salmonella. Thegenetic construct also includes, in addition to the encoding nucleicacid molecule, elements that allow expression, such as a promoter andregulatory sequences. The expression vectors may contain transcriptionalcontrol sequences that control transcriptional initiation, such aspromoter, enhancer, operator, and repressor sequences. A variety oftranscriptional control sequences are well known to those in the art.The expression vector can also include a translation regulatory sequence(e.g., an untranslated 5′ sequence, an untranslated 3′ sequence, or aninternal ribosome entry site). The vector can be capable of autonomousreplication or it can integrate into host DNA to ensure stability duringpeptide production.

The protein coding sequence that includes a peptide described herein canalso be fused to a nucleic acid encoding a peptide affinity tag, e.g.,glutathione S-transferase (GST), maltose E binding protein, protein A,FLAG tag, hexa-histidine (SEQ ID NO: 26), myc tag or the influenza HAtag, in order to facilitate purification. The affinity tag or reporterfusion joins the reading frame of the peptide of interest to the readingframe of the gene encoding the affinity tag such that a translationalfusion is generated. Expression of the fusion gene results intranslation of a single peptide that includes both the peptide ofinterest and the affinity tag. In some instances where affinity tags areutilized, DNA sequence encoding a protease recognition site will befused between the reading frames for the affinity tag and the peptide ofinterest.

Genetic constructs and methods suitable for production of immature andmature forms of the peptides and variants described herein in proteinexpression systems other than bacteria, and well known to those skilledin the art, can also be used to produce peptides in a biological system.

In other embodiments, peptides containing amino acids not normallyincorporated by the translation machinery and described above (e.g.—β-carboxylated Asp, γ-carboxylated Glu, Asu, Aad and Apm) may berecombinantly produced by tRNA modification methods. Methods formodifying tRNA including, but not limited to, modifying the anti-codon,the amino acid attachment site, and/or the accepter stem to allowincorporation of unnatural and/or arbitrary amino acids are known in theart (Biochem. Biophys. Res. Comm. (2008) 372: 480-485; Chem. Biol.(2009) 16:323-36; Nat. Methods (2007) 4:239-44; Nat. Rev. Mol. CellBiol. (2006) 7:775-82; Methods (2005) 36:227-238; Methods (2005)36:270-278; Annu. Rev. Biochem. (2004) 73:147-176; Nuc. Acids Res.(2004) 32:6200-6211; Proc. Natl. Acad. Sci. USA (2003) 100:6353-6357;Royal Soc. Chem. (2004) 33:422-430).

In some embodiments, peptides may be chemically produced. Peptides canbe synthesized by a number of different methods including solution andsolid phase synthesis using traditional BOC or FMOC protection. Forexample, the peptide can be synthesized on 2-Chlorotrityl or Wang resinusing consecutive amino acid couplings. The following protecting groupscan be used: Fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl(alpha-amino groups, N-terminus); trityl or tert-butyl (thiol groups ofCys); tert-butyl (γ-carboxyl of glutamic acid and the hydroxyl group ofthreonine, if present); and trityl (β-amid function of the asparagineside chain and the phenolic group of tyrosine, if present). Coupling canbe effected with DIC and HOBt in the presence of a tertiary amine, andthe peptide can be deprotected and cleaved from the solid support inusing cocktail K (trifluoroacetic acid 81%, phenol 5%, thioanisole 5%,1,2-ethanedithiol 2.5%, water 3%, dimethylsulphide 2%, ammonium iodide1.5% w/w). After removal of trifluoroacetic acid and other volatiles thepeptide can be precipitated using an organic solvent . Disulfide bondsbetween Cys residues can be formed using dimethyl sulfoxide (Tam et al.(1991) J. Am. Chem. Soc. 113:6657-62) or using an air oxidationstrategy. The resulting peptide can be purified by reverse-phasechromatography and lyophilized.

These peptides can be made, isolated or used either in form of the baseor as pharmaceutically acceptable salts thereof. Examples of saltsinclude, without limitation, acetate, chloride, sulfate and phosphatesalts of the peptide.

In some embodiments, the linaclotide composition comprises linaclotideor a pharmaceutically acceptable salt and a two or more peptidesselected from:

i. a peptide or a pharmaceutically acceptable salt thereof, wherein thepeptide comprises the amino acid structure of (SEQ ID NO: 2):

ii. a peptide or a pharmaceutically acceptable salt thereof, wherein thepeptide comprises an amino acid structure of (SEQ ID NO: 1):

andiii. a peptide or a pharmaceutically acceptable salt thereof, whereinthe peptide comprises the amino acid sequence Cys Cys Glu Tyr Cys CysAsn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 25), wherein at least onecarboxyl group of the peptide is an alkyl ester having the formula(—COOR) in which R is a C₁₋₆ alkyl.

In other embodiments, the linaclotide composition comprises linaclotideor a pharmaceutically acceptable salt thereof, one or more formaldehydescavenger compounds, and a peptide or a pharmaceutically acceptable saltthereof, wherein the peptide comprises an amino acid structure offormula (I) (SEQ ID NO: 3):

wherein the linaclotide composition contains up to 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, or 90% by weight of the peptide of formula (I) ascompared to the weight of linaclotide. In some preferred embodiments,the linaclotide composition contains up to 8% by weight of the peptideof formula (I) as compared to the weight of linaclotide. In somepreferred embodiments, the linaclotide composition contains up to 7% byweight of the peptide of formula (I) as compared to the weight oflinaclotide. In some preferred embodiments, the linaclotide compositioncontains up to 6% by weight of the peptide of formula (I) as compared tothe weight of linaclotide. In some preferred embodiments, thelinaclotide composition contains up to 5% by weight of the peptide offormula (I)as compared to the weight of linaclotide. In some preferredembodiments, the linaclotide composition contains up to 4% by weight ofthe peptide of formula (I) as compared to the weight of linaclotide. Insome preferred embodiments, the linaclotide composition contains up to3% by weight of the peptide of formula (I) as compared to the weight oflinaclotide. In some preferred embodiments, the linaclotide compositioncontains up to 2% by weight of the peptide of formula (I) as compared tothe weight of linaclotide. In some preferred embodiments, thelinaclotide composition contains up to 1% by weight of the peptide offormula (I) as compared to the weight of linaclotide. In some preferredembodiments, the linaclotide composition contains up to 0.75% by weightof the peptide of formula (I) as compared to the weight of linaclotide.In some preferred embodiments, the linaclotide composition contains upto 0.5% by weight of the peptide of formula (I) as compared to theweight of linaclotide.

In some preferred embodiments, the linaclotide composition contains oneor more formaldehyde scavenger compounds and an oral pharmaceuticaldosage form that comprises linaclotide and the peptide of formula (I)wherein the peptide of formula (I) is present in the oral pharmaceuticaldosage form in an amount of about 0.001 to about 2 wt. % relative to thetotal weight of the dosage form.

In some preferred embodiments, the linaclotide composition contains oneor more formaldehyde scavenger compounds and an oral pharmaceuticaldosage form that comprises linaclotide and the peptide of formula (I)wherein the peptide of formula (I) is present in the oral pharmaceuticaldosage form in an amount of about 0.001 to about 1 wt. % relative to thetotal weight of the dosage form.

In some preferred embodiments, the linaclotide composition contains oneor more formaldehyde scavenger compounds and an oral pharmaceuticaldosage form that comprises linaclotide and the peptide of formula (I)wherein the peptide of formula (I) is present in the oral pharmaceuticaldosage form in an amount of about 0.001 to about 0.75 wt. % relative tothe total weight of the dosage form.

In some preferred embodiments, the linaclotide composition contains oneor more formaldehyde scavenger compounds and an oral pharmaceuticaldosage form that comprises linaclotide and the peptide of formula (I)wherein the peptide of formula (I) is present in the oral pharmaceuticaldosage form in an amount of about 0.001 to about 0.5 wt. % relative tothe total weight of the dosage form.

In some embodiments, the imidazolidinone derivative of linaclotidecomprises up to about 15% by weight of the linaclotide composition, upto about 10% by weight of the linaclotide composition, up to about 7% byweight of the linaclotide composition or up to about 5% by weight of thelinaclotide composition. In other exemplary embodiments, theimidazolidinone derivative of linaclotide comprises from about 0.01% toabout 15% by weight of the linaclotide composition, about 0.05% to about10% by weight of the linaclotide composition, about 0.05% to about 7% byweight of the linaclotide composition or about 0.05% to about 5% byweight of the linaclotide composition.

In other embodiments, the linaclotide composition comprises linaclotideand a peptide or a pharmaceutically acceptable salt thereof, wherein thepeptide comprises an amino acid structure of (SEQ ID NO: 13):

wherein the peptide or pharmaceutically acceptable salt thereofcomprises up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weightcompared to the weight of linaclotide.

In further embodiments, the linaclotide composition compriseslinaclotide and a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises an amino acid structure of (SEQ ID NO:13):

wherein the peptide or pharmaceutically acceptable salt thereofcomprises up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight comparedto the weight of linaclotide.

In some embodiments, the Tyr₁₄-ethyl ester peptide comprises up to about15% by weight of the linaclotide composition, up to about 10% by weightof the linaclotide composition, up to about 7% by weight of thelinaclotide composition or up to about 5% by weight of the linaclotidecomposition. In other exemplary embodiments, the Tyr₁₄-ethyl estercomprises from about 0.01% to about 15% by weight of the linaclotidecomposition, about 0.05% to about 10% by weight of the linaclotidecomposition, about 0.05% to about 7% by weight of the linaclotidecomposition or about 0.05% to about 5% by weight of the linaclotidecomposition.

In other embodiments, the linaclotide composition comprising linaclotideand a peptide or a pharmaceutically acceptable salt thereof, wherein thepeptide comprises an amino acid structure of (SEQ ID NO: 10):

wherein the peptide or pharmaceutically acceptable salt thereofcomprises up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weightcompared to the weight of linaclotide.

In further embodiments, the linaclotide composition compriseslinaclotide and a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises an amino acid structure of (SEQ ID NO:10):

wherein the peptide or pharmaceutically acceptable salt thereofcomprises up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight comparedto the weight of linaclotide.

In some embodiments, the Glu3-ethyl ester peptide comprises up to about15% by weight of the linaclotide composition, up to about 10% by weightof the linaclotide composition, up to about 7% by weight of thelinaclotide composition or up to about 5% by weight of the linaclotidecomposition. In other exemplary embodiments, the Glu3-ethyl estercomprises from about 0.01% to about 15% by weight of the linaclotidecomposition, about 0.05% to about 10% by weight of the linaclotidecomposition, about 0.05% to about 7% by weight of the linaclotidecomposition or about 0.05% to about 5% by weight of the linaclotidecomposition.

In some embodiments, the linaclotide composition comprises a peptide orpharmaceutically acceptable salt, wherein the peptide consists of theamino acid structure of (SEQ ID NO: 3):

In other embodiments, the linaclotide composition consists essentiallyof a peptide or pharmaceutically acceptable salt thereof, wherein thepeptide comprises the amino acid structure of (SEQ ID NO: 3):

In other embodiments, the linaclotide composition consists linaclotide,a sterically hindered primary amine, a divalent metal cation, aformaldehyde scavenger compound, and one or more of the peptides havingthe structures of formula (VI) and (VII) or pharmaceutically acceptablesalt(s) thereof, wherein the linaclotide composition contains a totalcombined amount of the peptides of formula (VI) and (VII) up to 3%, 2%,1%, 0.75%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to0.01% by weight as compared to the weight of linaclotide.

In other embodiments, the linaclotide composition consists linaclotide,a sterically hindered primary amine, a divalent metal cation, aformaldehyde scavenger compound, and a peptide having the structure offormula (IX) or a pharmaceutically acceptable salt thereof (formula IXdisclosed as SEQ ID NO: 20):

wherein the linaclotide composition contains up to 3%, 2%, 1%, 0.75%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to 0.01% byweight of the peptide of formula (IX) as compared to the weight oflinaclotide.

In other embodiments, the linaclotide composition consists linaclotide,a sterically hindered primary amine, a divalent metal cation, aformaldehyde scavenger compound, and a peptide having the structure offormula (X) or a pharmaceutically acceptable salt thereof (formula Xdisclosed as SEQ ID NO: 21):

wherein the linaclotide composition contains up to 3%, 2%, 1%, 0.75%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to 0.01% byweight of the peptide of formula (X) as compared to the weight oflinaclotide.

In other embodiments, the linaclotide composition consists linaclotide,a sterically hindered primary amine, a divalent metal cation, aformaldehyde scavenger compound, and a peptide having the structure offormula (XI) or a pharmaceutically acceptable salt thereof (formula XIdisclosed as SEQ ID NO: 22):

wherein the linaclotide composition contains up to 3%, 2%, 1%, 0.75%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to 0.01% byweight of the peptide of formula (XI) as compared to the weight oflinaclotide.

In other embodiments, the linaclotide composition consists linaclotide,a sterically hindered primary amine, a divalent metal cation, aformaldehyde scavenger compound, and a peptide having the structure offormula (XII) or a pharmaceutically acceptable salt thereof (formula XIIdisclosed as SEQ ID NO: 23):

wherein the linaclotide composition contains up to 3%, 2%, 1%, 0.75%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to 0.01% byweight of the peptide of formula (XII) as compared to the weight oflinaclotide.

In other embodiments, the linaclotide composition consists linaclotide,a sterically hindered primary amine, a divalent metal cation, aformaldehyde scavenger compound, and a peptide having the structure offormula (XIII) or a pharmaceutically acceptable salt thereof (formulaXIII disclosed as SEQ ID NO: 24):

wherein the linaclotide composition contains up to 3%, 2%, 1%, 0.75%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to 0.01% byweight of the peptide of formula (XIII) as compared to the weight oflinaclotide.

In other embodiments, the linaclotide composition consists linaclotide,a sterically hindered primary amine, a divalent metal cation, aformaldehyde scavenger compound, and a peptide having the structure offormula (XIV) or a pharmaceutically acceptable salt thereof (formula XIVdisclosed as SEQ ID NO: 10):

wherein the linaclotide composition contains up to 3%, 2%, 1%, 0.75%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to 0.01% byweight of the peptide of formula (XIV) as compared to the weight oflinaclotide.

In other embodiments, the linaclotide composition consists linaclotide,a sterically hindered primary amine, a divalent metal cation, aformaldehyde scavenger compound, and a peptide having the structure offormula (XV) or a pharmaceutically acceptable salt thereof (formula XVdisclosed as SEQ ID NO: 13):

wherein the linaclotide composition contains up to 3%, 2%, 1%, 0.75%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to 0.01% byweight of the peptide of formula (XV) as compared to the weight oflinaclotide.

Methods of Treatment

In various embodiments, a patient diagnosed with a gastrointestinaldisorder is provided with the linaclotide composition described herein,wherein the gastrointestinal disorder is selected from the groupconsisting of irritable bowel syndrome (IBS) (such asconstipation-predominant IBS), constipation (such as chronic idiopathicconstipation), a functional gastrointestinal disorder, gastroesophagealreflux disease, functional heartburn, dyspepsia, visceral pain,gastroparesis, chronic intestinal pseudo-obstruction, colonicpseudo-obstruction, Crohn's disease, ulcerative colitis, andinflammatory bowel disease.

In a further embodiment, the gastrointestinal disorder is constipation.The constipation can be chronic idiopathic constipation, idiopathicconstipation, due to post-operative ileus, or caused by opiate use.Clinically accepted criteria that define constipation include thefrequency of bowel movements, the consistency of feces and the ease ofbowel movement. One common definition of constipation is less than threebowel movements per week. Other definitions include abnormally hardstools or defecation that requires excessive straining (Schiller 2001,Aliment Pharmacol Ther 15:749-763). Constipation may be idiopathic(functional constipation or slow transit constipation) or secondary toother causes including neurologic, metabolic or endocrine disorders.These disorders include diabetes mellitus, hypothyroidism,hyperthyroidism, hypocalcaemia, Multiple Sclerosis, Parkinson's disease,spinal cord lesions, Neurofibromatosis, autonomic neuropathy, Chagasdisease, Hirschsprung's disease and Cystic fibrosis. Constipation mayalso be the result of surgery (postoperative ileus) or due to the use ofdrugs such as analgesics (like opioids), antihypertensives,anticonvulsants, antidepressants, antispasmodics and antipsychotics.

In other embodiments, the gastrointestinal disorder is irritable bowelsyndrome (IBS). The irritable bowel syndrome can beconstipation-predominant irritable bowel syndrome (c-IBS),diarrhea-predominant irritable bowel syndrome (d-IBS) or alternatingbetween the two irritable bowel syndromes (a-IBS).

In other embodiments, the gastrointestinal disorder is dyspepsia.

In other embodiments, the gastrointestinal disorder is gastroparesis.The gastroparesis can be selected from idiopathic, diabetic orpost-surgical gastroparesis.

In still other embodiments, the gastrointestinal disorder is chronicintestinal pseudo obstruction.

In other embodiments, the gastrointestinal disorder is Crohn's disease.

In some embodiments, the gastrointestinal disorder is ulcerativecolitis.

In some embodiments, the gastrointestinal disorder is inflammatory boweldisease.

In still another embodiment, the invention features a method fortreating a gastrointestinal disorder comprising providing a patientdiagnosed with IBS-C or CC with the linaclotide composition describedherein.

It has been discovered in some embodiments, that linaclotide is only tobe administered to patients without known or suspected mechanicalgastrointestinal obstruction. In this regard, prior to administering orproviding the linaclotide composition to patients diagnosied with a GIdisorder, the patient is evaluated for symptoms suggestive of mechanicalgastrointestinal obstruction prior to initiating treatment.

Moreover, in some embodiments, the linaclotide composition is providedto patients diagnosed with a GI disorder and not currently or frequentlyexperiencing diarrhea.

In some embodiments, the method comprises after administering orproviding the linaclotide composition to a patient, monitoring thepatient for diarrhea, flatulence, abdominal pain, abdominal distension,Defecation urgency, dyspepsia, gastroesophageal reflux disease, upperabdominal pain, vomiting; viral gastroenteritis; muscle strain;dizziness; sinus congestion, fecal incontinence, dehydration and/orheadache, and optionally decreasing the linaclotide dose administered topatients experiencing one or more of these adverse reactions to theinitial dosage.

In some embodiments, the method comprises after administering orproviding the linaclotide composition to a patient, periodically assessthe need for continued treatment with linaclotide.

In some embodiments, the method comprises after administering orproviding the linaclotide composition to a patient, determining that thepatient receiving the linaclotide composition is experiencing severe orintolerable diarrhea, and reducing the linaclotide dose or suspendingtreatment.

In some embodiments, the method comprises diagnosing the patient withIBS-C or CC, administering or providing the linaclotide composition tothe patient and counseling the patient that improvement of bowelsymptoms should occur within the first week of treatment, butimprovement of abdominal symptoms may take longer.

In some embodiments, the method comprises periodically assessing theneed for continued treatment with the linaclotide composition.

In this regard, the efficacy of oral dosage forms comprising 290 mg forthe management of IBS-C was established in two double-blind,placebo-controlled, randomized, multicenter studies in adult patients. Atotal of 800 patients in Study 1 and 804 patients in Study 2 (overallmean age of 43.9 years [range 18-87 years with 5.3%≥65 years of age],90.1% female, 77.4% white, 18.8% black, and 12.0% Hispanic) receivedtreatment with LINZESS 290 mcg or placebo once daily and were evaluatedfor efficacy. All patients met Rome II criteria for IBS and wererequired to report a mean abdominal pain score of ≥3 on a 0-to-10-pointnumeric rating scale, <3 complete spontaneous bowel movements (CSBMs; aCSBM is a spontaneous bowel movement [SBM] that is associated with asense of complete evacuation; an SBM is a bowel movement occurring inthe absence of laxative use), and ≤5 SBMs per week during a 2-weekbaseline period. The study designs were identical through the first 12weeks, and thereafter differed only in that Study 1 included a 4-weekrandomized withdrawal (RW) period, and Study 2 continued for 14additional weeks (total of 26 weeks) of double-blinded treatment .

Efficacy of the oral dosage form of linaclotide was assessed usingresponder and change-from-baseline endpoints. Results for endpoints werebased on information provided daily by patients. An abdominal painresponder was a patient who had ≥30% reduction in mean abdominal painfrom baseline in a given week for ≥6 out of 12 weeks of the treatmentperiod. A CSBM responder was a patient who had an increase of ≥1 CSBMfrom baseline in a given week for ≥6 out of 12 weeks of the treatmentperiod. To be a combined responder, a patient had to meet both abdominalpain and CSBM weekly responder criteria in the same week for ≥6 out of12 weeks of the treatment period. The efficacy results are shown inTable 2. In both studies, the proportion of patients who were respondersto the oral dosage form of 290 mcg linaclotide was statisticallysignificantly higher than with placebo.

For change-from-baseline endpoints, patients who received the oraldosage form of 290 mcg linaclotide across the 2 studies hadstatistically significantly (p<0.0001) greater improvements comparedwith patients receiving placebo in abdominal symptoms, includingabdominal pain, abdominal discomfort, and bloating; and bowel function,including stool frequency (CSBM and SBM) and consistency (i.e., hardnessof stool), as well as straining. Sixty-seven percent of the patients hadan SBM within 24 hours of taking their first dose versus 42% of placebopatients (p<0.0001).

The proportions of patients who met response criteria of increasinglevels of symptom improvement compared to baseline (i.e., decreases of≥0%, ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, and ≥60% in abdominal pain andincreases of ≥0, ≥1, ≥2, ≥3, ≥4, ≥5, and ≥6 CSBMs per week) over 12weeks of treatment were analyzed. At each level, a statisticallysignificantly greater proportion of patients treated with the oraldosage form of 290 mcg linaclotide met the response criterion comparedto placebo patients. Moreover, the oral dosage form of 290 mcglinaclotide demonstrated a statistically significant separation fromplacebo that was present at the first week and sustained across the 26weeks of the treatment period (p<0.001 at all time points during thetreatment period). Similar results for improvement in CSBM frequencywere demonstrated throughout the 26-week treatment period. Maximumeffect on CSBM frequency occurred by Week 1, but the effect on abdominalpain continued to increase over the first 6 to 8 weeks. During the4-week RW period in Study 2 when treatment with the oral dosage form of290 mcg linaclotide was discontinued, bowel symptoms returned towardbaseline within the first week with no evidence of rebound worseningcompared to baseline; abdominal symptoms also returned toward baselinewith no evidence of rebound.

The efficacy of the oral dosage form of 290 mcg linaclotide for themanagement of CC was established in two double-blind,placebo-controlled, randomized, multicenter studies in adult patients. Atotal of 642 patients in Study 3 and 630 patients in Study 4 (overallmean age of 47.8 years [range 18-85 years with 12.1%≥65 years of age],88.9% female, 76.2% white, 21.5% black, Hispanic 10.0%) receivedtreatment with the oral dosage form of 145 or 290 mcg linaclotide, orplacebo once daily and were evaluated for efficacy. All patients metRome II criteria for CC and were required to report <3 CSBMs and ≤6 SBMsper week during a 2-week baseline period. Patients were excluded if theymet criteria for IBS. The study designs differed only in that Study 3had a 4-week RW period following the 12-week treatment period.

Efficacy of the oral dosage form of 145 or 290 mcg linaclotide wasassessed using responder and change-from-baseline endpoints. Results forendpoints were based on information provided daily by patients. A CSBMresponder was defined differently in the CC studies than it was in theIBS-C studies. A CSBM responder in the CC studies was a patient who had≥3 CSBMs and an increase of ≥1 CSBM from baseline in a given week for ≥9out 12 weeks of the treatment period. both studies, the proportion ofpatients who were CSBM responders was statistically significantlygreater with each dose of LINZESS (145 and 290 mcg) than with placebo.

For change-from-baseline endpoints, patients who received either dose ofthe oral dosage form of linaclotide across the 2 studies hadstatistically significantly (p<0.0001) greater improvements comparedwith patients receiving placebo in abdominal discomfort, bloating, stoolfrequency (CSBM and SBM), stool consistency (i.e., hardness of stool),and straining. Sixty-seven percent and 57% of linaclotide 145 and 290mcg patients, respectively, had an SBM within 24 hours of taking theirfirst dose versus 39% of placebo patients (p<0.0001 for both dosesversus placebo).

The proportions of patients who met response criteria of increasinglevels of stool frequency compared to baseline (i.e., increases of >0,≥1, ≥2, ≥3, ≥4, ≥5, and ≥6 CSBMs per week) over 12 weeks of treatmentwere analyzed. At each level, a statistically significantly greaterproportion of patients treated with either dose of the oral dosage formof linaclotide met the response criterion compared with placebopatients.

For CSBM and SBM frequency, each dose of LINZESS (145 and 290 mcg)demonstrated a statistically significant separation from placebo thatwas present at the first week and sustained across the 12 weeks of thetreatment period (p<0.001 for each dose vs. placebo at all time points).During the 4-week RW period in Study 3 when LINZESS treatment wasdiscontinued, bowel function, including CSBMs and SBMs, returned towardbaseline within the first week with no evidence of rebound worsening(see FIG. 3).

In some preferred embodiments, the linaclotide composition is stored upto 25° C. (77° F.); excursions permitted between 15° C. and 30° C. (59°F. and 86° F.) [see USP Controlled Room Temperature]. In someembodiments, the linaclotide composition is stored is a low moistureenvironment.

As used herein, unless otherwise indicated, the phrase “consisting of”when used in reference to the linaclotide composition or a singlecomponent of the composition means that the linaclotide composition orsingle component defined by the phrase contains no other components thanthose specified but may contain additional components that are unrelatedto the invention and/or impurities ordinarily associated with therecited steps or components.

Dosage and Excipients

The linaclotide composition may any suitable oral pharmaceutical dosageform (e.g., capsules) containing any suitable therapeutic dosage oflinaclotide. In some embodiments, the linaclotide composition comprisescapsules/tablets that comprise 290 mcg which are to be taken orally oncedaily on an empty stomach, such as for treating IBS-c (MS withconstipation. In some embodiments, the linaclotide composition comprisescapsules/tablets that comprise 145 or 290 mcg which are to be takenorally once daily on an empty stomach for treating chronic idiopathicconstipation.

The linaclotide composition can include additional ingredients orexcipient. In certain embodiments, one or more therapeutic agents of thedosage unit may exist in an extended or control release formulation andadditional therapeutic agents may not exist in extended releaseformulation. For example, a peptide or agonist described herein mayexist in a controlled release formulation or extended releaseformulation in the same dosage unit with another agent that may or maynot be in either a controlled release or extended release formulation.Thus, in certain embodiments, it may be desirable to provide for theimmediate release of one or more of the agents described herein, and thecontrolled release of one or more other agents.

The linaclotide composition can comprise any pharmaceutically tolerablecarrier or medium, e.g. solvents, dispersants, coatings, absorptionpromoting agents, controlled release agents, and one or more inertexcipients (which include starches, polyols, granulating agents,microcrystalline cellulose (e.g. celphere, Celphere beads®), diluents,lubricants, binders, disintegrating agents, and the like), etc. Ifdesired, tablet dosages of the disclosed compositions may be coated bystandard aqueous or nonaqueous techniques.

Examples of excipients for use as the pharmaceutically acceptablecarriers and the pharmaceutically acceptable inert carriers and theaforementioned additional ingredients include, but are not limited tobinders, fillers, disintegrants, lubricants, anti-microbial agents, andcoating agents.

As used herein, the term “binder” refers to any pharmaceuticallyacceptable binder that may be used in the practice of the invention.Examples of pharmaceutically acceptable binders include, withoutlimitation, a starch (e.g., corn starch, potato starch andpre-gelatinized starch (e.g., STARCH 1500® and STARCH 1500 LM®, sold byColorcon, Ltd.) and other starches), maltodextrin, gelatin, natural andsynthetic gums such as acacia, powdered tragacanth, guar gum, celluloseand its derivatives (e.g., methylcellulose, hydroxyethyl cellulose,hydroxyethyl methylcellulose, hydroxypropyl cellulose and hydroxypropylmethylcellulose (hypromellose), ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose,carboxymethylcellulose, microcrystalline cellulose (e.g. AVICEL™ suchas, AVICEL-PH-101™, -103™ and 105™, sold by FMC Corporation, MarcusHook, PA, USA)), polyvinyl alcohol, polyvinyl pyrrolidone (e.g.,polyvinyl pyrrolidone K30), and mixtures thereof.

As used herein, the term “filler” refers to any pharmaceuticallyacceptable filler that may be used in the practice of the invention.Examples of pharmaceutically acceptable fillers include, withoutlimitation, talc, calcium carbonate (e.g., granules or powder), dibasiccalcium phosphate, tribasic calcium phosphate, calcium sulfate (e.g.,granules or powder), microcrystalline cellulose (e.g., Avicel PH101 orCelphere CP-305), powdered cellulose, dextrates, kaolin, mannitol,silicic acid, sorbitol, starch (e.g., Starch 1500), pre-gelatinizedstarch, lactose, glucose, fructose, galactose, trehalose, sucrose,maltose, isomalt, raffinose, maltitol, melezitose, stachyose, lactitol,palatinite, xylitol, myoinositol, and mixtures thereof

Examples of pharmaceutically acceptable fillers that may be particularlyused for coating the peptides include, without limitation, talc,microcrystalline cellulose (e.g., Avicel PH101 or Celphere CP-305),powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol,starch, pre-gelatinized starch, lactose, glucose, fructose, galactose,trehalose, sucrose, maltose, isomalt, dibasic calcium phosphate,raffinose, maltitol, melezitose, stachyose, lactitol, palatinite,xylitol, mannitol, myoinositol, and mixtures thereof.

As used herein, the term “additives” refers to any pharmaceuticallyacceptable additive. Pharmaceutically acceptable additives include,without limitation, disintegrants, dispersing additives, lubricants,glidants, antioxidants, coating additives, diluents, surfactants,flavoring additives, humectants, absorption promoting additives,controlled release additives, anti-caking additives, anti-microbialagents (e.g., preservatives), colorants, desiccants, plasticizers anddyes. As used herein, an “excipient” is any pharmaceutically acceptableadditive, filler, binder or agent.

The linaclotide composition may also optionally include othertherapeutic ingredients, anti-caking agents, preservatives, sweeteningagents, colorants, flavors, desiccants, plasticizers, dyes, glidants,anti-adherents, anti-static agents, surfactants (wetting agents),anti-oxidants, film-coating agents, and the like. Any such optionalingredient must be compatible with the compound described herein toinsure the stability of the formulation. The composition may containother additives as needed, including for example lactose, glucose,fructose, galactose, trehalose, sucrose, maltose, raffinose, maltitol,melezitose, stachyose, lactitol, palatinite, starch, xylitol, mannitol,myoinositol, and the like, and hydrates thereof, and amino acids, forexample alanine, glycine and betaine, and peptides and proteins, forexample albumen.

The linaclotide composition can include, for example, various additionalsolvents, dispersants, coatings, absorption promoting additives,controlled release additives, and one or more inert additives (whichinclude, for example, starches, polyols, granulating additives,microcrystalline cellulose, diluents, lubricants, binders,disintegrating additives, and the like), etc. If desired, tablet dosagesof the disclosed compositions may be coated by standard aqueous ornon-aqueous techniques. Compositions can also include, for example,anti-caking additives, preservatives, sweetening additives, colorants,flavors, desiccants, plasticizers, dyes, and the like.

Suitable disintegrants include, for example, agar-agar, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, povidone, polacrilin potassium, sodium starch glycolate,potato or tapioca starch, other starches, pre-gelatinized starch, clays,other algins, other celluloses, gums, and mixtures thereof.

Suitable lubricants include, for example, calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil), zincstearate, ethyl oleate, ethyl laurate, agar, syloid silica gel (AEROSIL200, W.R. Grace Co., Baltimore, Md. USA), a coagulated aerosol ofsynthetic silica (Evonik Degussa Co., Plano, Tex. USA), a pyrogenicsilicon dioxide (CAB-O-SIL, Cabot Co., Boston, Mass. USA), and mixturesthereof.

Suitable glidants include, for example, leucine, colloidal silicondioxide, magnesium trisilicate, powdered cellulose, starch, talc, andtribasic calcium phosphate.

Suitable anti-caking additives include, for example, calcium silicate,magnesium silicate, silicon dioxide, colloidal silicon dioxide, talc,and mixtures thereof.

Suitable anti-microbial additives that may be used, e.g., as apreservative for the peptides compositions, include, for example,benzalkonium chloride, benzethonium chloride, benzoic acid, benzylalcohol, butyl paraben, cetylpyridinium chloride, cresol, chlorobutanol,dehydroacetic acid, ethylparaben, methylparaben, phenol, phenylethylalcohol, phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate,potassium sorbate, propylparaben, sodium benzoate, sodiumdehydroacetate, sodium propionate, sorbic acid, thimersol, thymo, andmixtures thereof.

Suitable antioxidants include, for example, BHA (butylatedhydroxyanisole), BHT (butylated hydroxytoluene), vitamin E, propylgallate, ascorbic acid and salts or esters thereof, tocopherol andesters thereof, alpha-lipoic acid and beta-carotene.

Suitable coating additives include, for example, sodium carboxymethylcellulose, cellulose acetate phthalate, ethylcellulose, gelatin,pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl methyl cellulose phthalate,methylcellulose, polyethylene glycol, polyvinyl acetate phthalate,shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline wax,and mixtures thereof. Suitable protective coatings include Aquacoat(e.g. Aquacoat Ethylcellulose Aquaeous Dispersion, 15% w/w, FMCBiopolymer, ECD-30), Eudragit (e.g. Eudragit E PO PE-EL, Roehm PharmaPolymers) and Opadry (e.g Opadry AMB dispersion, 20% w/w, Colorcon).

In certain embodiments, suitable additives for the peptides compositioninclude one or more of sucrose, talc, magnesium stearate, crospovidoneor BHA.

The compositions of the present invention can also include otherexcipients, agents, and categories thereof including but not limited toL-histidine, Pluronic®, Poloxamers (such as Lutrol® and Poloxamer 188),ascorbic acid, glutathione, permeability enhancers (e.g. lipids, sodiumcholate, acylcarnitine, salicylates, mixed bile salts, fatty acidmicelles, chelators, fatty acid, surfactants, medium chain glycerides),protease inhibitors (e.g. soybean trypsin inhibitor, organic acids), pHlowering agents and absorption enhancers effective to promotebioavailability (including but not limited to those described in U.S.Pat. Nos. 6,086,918 and 5,912,014), materials for chewable tablets (likedextrose, fructose, lactose monohydrate, lactose and aspartame, lactoseand cellulose, maltodextrin, maltose, mannitol, microcrystallinecellulose and guar gum, sorbitol crystalline); parenterals (likemannitol and povidone); plasticizers (like dibutyl sebacate,plasticizers for coatings, polyvinylacetate phthalate); powderlubricants (like glyceryl behenate); soft gelatin capsules (likesorbitol special solution); spheres for coating (like sugar spheres);spheronization agents (like glyceryl behenate and microcrystallinecellulose); suspending/gelling agents (like carrageenan, gellan gum,mannitol, microcrystalline cellulose, povidone, sodium starch glycolate,xanthan gum); sweeteners (like aspartame, aspartame and lactose,dextrose, fructose, honey, maltodextrin, maltose, mannitol, molasses,sorbitol crystalline, sorbitol special solution, sucrose); wetgranulation agents (like calcium carbonate, lactose anhydrous, lactosemonohydrate, maltodextrin, mannitol, microcrystalline cellulose,povidone, starch), caramel, carboxymethylcellulose sodium, cherry creamflavor and cherry flavor, citric acid anhydrous, citric acid,confectioner's sugar, D&C Red No. 33, D&C Yellow #10 Aluminum Lake,disodium edetate, ethyl alcohol 15%, FD& C Yellow No. 6 aluminum lake,FD&C Blue #1 Aluminum Lake, FD&C Blue No. 1, FD&C blue no. 2 aluminumlake, FD&C Green No.3, FD&C Red No. 40, FD&C Yellow No. 6 Aluminum Lake,FD&C Yellow No. 6, FD&C Yellow No.10, glycerol palmitostearate, glycerylmonostearate, indigo carmine, lecithin, manitol, methyl and propylparabens, mono ammonium glycyrrhizinate, natural and artificial orangeflavor, pharmaceutical glaze, poloxamer 188, Polydextrose, polysorbate20, polysorbate 80, polyvidone, pregelatinized corn starch,pregelatinized starch, red iron oxide, saccharin sodium, sodiumcarboxymethyl ether, sodium chloride, sodium citrate, sodium phosphate,strawberry flavor, synthetic black iron oxide, synthetic red iron oxide,titanium dioxide, and white wax.

In some embodiments, there is provided a pharmaceutical compositioncomprising a peptide described herein and one or more agents selectedfrom Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, Na⁺ or Al³⁺, a combination thereof, and/ora sterically hindered primary amine. In further embodiments, the agentis Mg²⁺, Ca²⁺ or Zn²⁺ or a combination thereof. In some embodiments, thedivalent metal cation is provided, without limitation, as magnesiumacetate, magnesium chloride, magnesium phosphate, magnesium sulfate,calcium acetate, calcium chloride, calcium phosphate, calcium sulfate,zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganeseacetate, manganese chloride, manganese phosphate, manganese sulfate,potassium acetate, potassium chloride, potassium phosphate, potassiumsulfate, sodium acetate, sodium chloride, sodium phosphate, sodiumsulfate, aluminum acetate, aluminum chloride, aluminum phosphate oraluminum sulfate. In further embodiments, the cation is provided asmagnesium chloride, calcium chloride, calcium phosphate, calciumsulfate, zinc acetate, manganese chloride, potassium chloride, sodiumchloride or aluminum chloride. In other embodiments, the cation isprovided as calcium chloride, magnesium chloride or zinc acetate.

In another embodiment, the agent is a sterically hindered primary amine.In a further embodiment, the sterically hindered primary amine is anamino acid. In yet a further embodiment, the amino acid is anaturally-occurring amino acid. In a still further embodiment, thenaturally-occurring amino acid is selected from the group consisting of:histidine, phenylalanine, alanine, glutamic acid, aspartic acid,glutamine, leucine, methionine, asparagine, tyrosine, threonine,isoleucine, tryptophan, glycine and valine; yet further, thenaturally-occurring amino acid is leucine, isoleucine, alanine ormethionine. In a still further embodiment, the naturally-occurring aminoacid is leucine. In another embodiment, the sterically hindered primaryamine is a non-naturally occurring amino acid (e.g., 1-aminocyclohexanecarboxylic acid). In a further embodiment, the sterically hinderedprimary amine is cyclohexylamine, 2-methylbutylamine or chitosan. Inanother embodiment, one or more sterically hindered primary amines maybe used in a composition.

In some cases, the sterically hindered primary amine has the formula:,wherein R₁, R₂ and R₃ are independently selected from: H, C(O)OH, C1-C6alkyl, C1-C6 alkylether, C1-C6 alkylthioether, C1-C6 alkyl carboxylicacid, C1-C6 alkyl carboxylamide and alkylaryl, wherein any group can besingly or multiply substituted with: halogen or amino, and provided thatno more than two of R₁, R₂ and R₃ are H. In another embodiment, no morethan one of R₁, R₂ and R₃ is H.

In other embodiments, there is provided a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, peptide, a cationselected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, Na⁺ or Al³⁺, or a mixture thereof,and a sterically hindered primary amine. In one embodiment, the cationis Mg²⁺, Ca²⁺ or Zn²⁺ or a mixture thereof. In a further embodiment, thelinaclotide composition further comprises a pharmaceutically acceptablebinder and/or a pharmaceutically acceptable glidant, lubricant oradditive that acts as both a glidant and lubricant and/or anantioxidant. In some embodiments, the linaclotide composition is appliedto a carrier. In some embodiments, the carrier is a filler.

In some cases the molar ratio of cation:sterically hindered primaryamine: peptide in the aqueous solution applied to the carrier is5-100:5-50:1. In some cases, the molar ratio of cation:stericallyhindered primary amine may be equal to or greater than 2:1 (e.g.,between 5:1 and 2:1). Thus, in some cases the molar ratio ofcation:sterically hindered primary amine: peptide applied to the carrieris 100:50:1, 100:30:1, 80:40:1, 80:30:1, 80:20:1, 60:30:1, 60:20:1,50:30:1, 50:20:1, 40:20:1, 20:20:1, 10:10:1, 10:5:1 or 5:10:1. In somepreferred embodiments, the molar ratio of divalent metalcation:sterically hindered primary amine:linaclotide is about57-63:28-32:1. When binder, e.g., methylcellulose, is present in theGC-C agonist peptide solution applied to the carrier it can be presentat 0.5%-2.5% by weight (e.g., 0.7%-1.7% or 0.7% - 1% or 1.5% or 0.7%).

In a further embodiment, the linaclotide composition further comprises apharmaceutically acceptable binder or additive, and/or apharmaceutically acceptable glidant, lubricant or additive that acts asboth a glidant and lubricant and/or an antioxidant.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of the active compound(s) with anacceptable pharmaceutical diluent or excipient, such as a sterileaqueous solution, to give a range of final concentrations, depending onthe intended use. The techniques of preparation are generally well knownin the art, as exemplified by Remington's Pharmaceutical Sciences (18thEdition, Mack Publishing Company, 1995).

As used herein, unless otherwise indicated, the phrase “consistingessentially of,” when used in reference to the linaclotide compositionor a single component of the composition means that the composition orsingle component defined by the phrase contains no active pharmaceuticalingredients other than those specified but that it may contain anyadditional inactive components or excipients or formaldehyde scavengercompound(s).

The term “linaclotide composition” is used herein, unless otherwiseindicated, to mean a composition that comprises linaclotide, excipientsnecessary for stabilizing, storing and delivering linaclotide asdescribed herein (e.g., a divalent metal cation and an amino acid, forexample, in a molar ratio of divalent metal cation:aminoacid:linaclotide of about 57-63:28-32:1) and optionally one or moreformaldehyde scavenger compounds in any suitable configuration orconformation. For example, in some embodiments, the linaclotidecomposition comprises an oral pharmaceutical dosage form (e.g., capsulesor tablets) that comprises linaclotide (in any desired dosage, such as145 μg or 290 μg), excipients necessary for stabilizing, storing anddelivering linaclotide as described herein (e.g., a divalent metalcation and an amino acid for example in a molar ratio of divalent metalcation:amino acid:linaclotide of about 57-63:28-32:1) and one or moreformaldehyde scavenger compounds. In other preferred embodiments, thelinaclotide composition comprises an oral pharmaceutical dosage form(e.g., capsules or tablets) that comprises linaclotide (in any desireddosage, such as 145 μg or 290 μg) and excipients necessary forstabilizing, storing and delivering linaclotide such as described herein(e.g., a divalent metal cation and an amino acid in a molar ratio ofdivalent metal cation:amino acid:linaclotide of about 57-63:28-32:1) andone or more separate formaldehyde scavenger compounds that are outsideof the oral dosage form (e.g., but in the same bottle, cannister, orcontainer).

The present invention has been described with reference to certainexemplary embodiments thereof. However, it will be readily apparent tothose skilled in the art that it is possible to embody the invention inspecific forms other than those of the exemplary embodiments describedabove. This may be done without departing from the spirit of theinvention. The exemplary embodiments are merely illustrative and shouldnot be considered restrictive in any way. The scope of the invention isdefined by the appended claims and their equivalents, rather than by thepreceding description.

EXAMPLES

The GC-C agonist peptides or pharmaceutically acceptable salts thereofas described herein were prepared by solid phase chemical synthesis andnatural folding (air oxidation) by American Peptide Company (Sunnyvale,Calif.). In some cases, the peptides were modified after synthesis asdescribed herein.

The Cys₁-IMD peptide was synthesized by mixing 4.6 g (3.0 mmol) oflinaclotide in 200 ml of EtOH. Formaldehyde at 37% (1.12 ml/5eq) wasadded to this mixture. The reaction mixture was incubated in a waterbath (45□C) for overnight. The following day the solvent was removed byrota-evaporation. The peptide was further purified through reverse-phasechromatography.

The Glu3-ethyl ester peptide was synthesized on a 20 mmolFmoc-Tyr(tBu)-Wang resin. Protecting groups used for amino acids are:t-Butyl group for Tyr and Thr, Trt group for Asn and Cys. The peptidechain was assembled on the resin by repetitive removal of the Fmocprotecting group and coupling of protected amino acid. DIC and HOBt wereused as coupling reagents and NMM was used as the base for thisreaction. 20% piperidine in DMF was used as de-Fmoc-reagent. Afterremoval of last Fmoc protecting group, resin was treated with cocktail Kfor 3 hours to cleave the peptide from resin and removal of the sidechain protecting groups. The eluted peptide was precipitated in coldether and dried. The dried peptide was dissolved in a mixture ofTFA/TIS/water (95:3:2v/v) in a ratio of 1 to 10 (g/v). This mixture wasstirred at room temperature for 1 hour. The isolated peptide was alsoprecipitated in cold ether, collected by filtration and dried under highvacuum.

The Tyr₁₄-ethyl ester peptide was synthesized by a fragment condensationmethod. Fragment A(Boc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-OH(SEQ ID NO: 27)) was prepared on 15mmol CTC resin using Fmoc chemistry.This peptide chain was also assembled on the resin by repetitive removalof the Fmoc protecting group and coupling of protected amino acid. DICand HOBt were used as coupling reagents and NMM was used as the base.20% piperidine in DMF was used as de-Fmoc-reagent. After removal of lastFmoc protecting group, Boc was coupled to protect the N-terminal aminogroup. The peptide resin was washed, dried, and treated with 1% TFA/DCMto cleave peptide from resin. Fragment B (Cys(Trt)-Tyr-OEt) was preparedfrom coupling of Fmoc-Cys(Trt)-OH and Tyr-OEt. HCl. The Fmoc group wasremoved by treating this di-peptide with 20% piperidine in DMF.

The Tyr₁₄-ethyl ester peptide was finally synthesized by coupling thetwo fragments in DMF. HBTU/HOBt/NMM was used as the coupling reagent forthis reaction. The protecting groups were removed by treating thepeptide with cocktail K for 2 hours. This peptide was precipitated incold ether and dried. The dried peptide was dissolved in a mixture ofTFA/TIS/water (95:3:2v/v) in a ratio of 1 to 10 (g/v). This mixture wasstirred at room temperature for 1 hour. The isolated peptide was againprecipitated in cold ether, collected by filtration and dried under highvacuum.

Example 1: cGMP Accumulation in T84 Cells for Analysis of GC-C Activity

For the cGMP assay, 4.5×10⁵ cells/mL of T84 cells were grown overnightin 24 well tissue culture plates. On next day, the T84 cells were washedtwice with 1 mL of DMEM (pH 7). After the second wash, the cells wereincubated with 450 μL of 1 mM isobutylmethylxanthine (IBMX) in pH 7buffer for 10 minutes at 37° C. to inhibit any phosphodiesteraseactivity. The peptides were then diluted in DMEM buffer (pH 7) to a 10×concentration. The peptide solution of 50 μL was diluted to a finalvolume of 500 μL with the T84 cells, bringing each peptide concentrationto lx. The peptides were tested in duplicate at 100 nM.

There was no peptide control used to determine endogenous levels ofcGMP. Peptides were incubated for 30 minutes at 37° C. After 30 minutes,the supernatants were removed and the cells were lysed with 0.1 M HCl.The cells were lysed for 30 minutes on ice. After 30 minutes, lysateswere pipetted off and placed into a 96 well HPLC plate and spun at10,000×G for 10 minutes to remove any cell debris. Supernatants from theprevious spin were removed and placed into a fresh 96 well HPLC plate.

cGMP concentrations were determined from each sample using the LC/MSconditions (Table 1 below) and calculated standard curve. EC₅₀ valueswere calculated from concentration-response curves generated withGraphPad Prism Software.

TABLE 1 LC/MS conditions MS: Thermo Quantum Ion Mode: ESI⁺ Scan Type:MRM Dwell Collision Retention Time Energy Tube Time Compound: Transition(msec) (V) Lens (min) cGMP 346 > 152 100 28 139 1.0 HPLC: AgilentTechnologies 1200 Series with CTC Analytics HTS PAL Column: ThermoHypersil Gold 2.1 × 50 mm 5 micron particle size Flow Rate: 400 uL/minColumn RT Temperature: Autosampler 6° C. Temperature: Injection 20 uLVolume: Mobile A = 98:2 Water:Acetonitrile + 0.1% Formic Acid Phases: B= 2:98 Water:Acetonitrile + 0.1% Formic Acid Time Gradient: (min) % A %B 0 100 0 0.3 30 70 2.00 30 70 2.01 100 0 4 100 0

Example 2: Relative Binding Affinity of Exemplary Peptides to the GC-CReceptor of T84 Cells

The relative binding affinities of linaclotide and Cys₁-IMD to theguanylate cyclase-C receptor (GC-C) were determined using acompetitive-binding assay in which the peptides competed with a knownGC-C agonist, porcine-derived heat-stable enterotoxin (pSTa), forbinding sites on cell-surface GC-C receptors on human colonic epithelial(T84) cells. The pSTa was radiolabeled with ¹²⁵I to enable measurementof its receptor binding. The competitive-binding assay was performed byadding various concentrations of each peptide (0.1 to 3,000 nM) to0.20mL reaction mixtures containing Dulbecco's modified Eagle's medium(DMEM), 0.5% bovine serum albumin (BSA), 2.0×10⁵ T84 cells, and 170 pM[¹²⁵I]STa (200,000 cpm). The data were used to construct competitiveradioligand-binding curves and determine the relative binding affinitiesof linaclotide and Cys₁-IMD, as measured by their IC₅₀ and K_(i) values.

Both linaclotide and Cys₁-IMD competitively inhibited the specificbinding of [¹²⁵I]-pSTa to cell-surface GC-C receptors on T84 cells.Their relative binding affinities, as measured by their inhibitionconstants (K), were as follows: Linaclotide K=3.9±1.6 nM and Cys₁-IMDK=1.4±0.5 nM (FIG. 3).

Example 3: cGMP Response in T84 Cells Induced by Exemplary Peptides

Linaclotide and Cys₁-IMD were tested for guanylate cyclase-C (GC-C)agonist activity in T84 cells as follows. In each well of a 96-wellplate, approximately 200,000 T84 cells/well was first incubated with 1mM 3-isobutyl-1-methylxanthine (IBMX) in 0.18 mL of Dulbecco's modifiedEagle's medium (DMEM) for 10 minutes at 37° C. Each peptide was dilutedto final concentrations ranging from 0.1 to 10,000 nM, and 0.02 mL ofeach dilution was added in duplicate to a 96-well plate containing theT84 cells, for a final volume of 0.20 mL per well. The peptide reactionswere incubated for 30 min at 37° C. Following the incubation, thesupernatants were removed and discarded and the cells were lysed withcold 0.1 M hydrochloric acid (HCl) for 30 min on ice. The cell debriswas removed by centrifugation and the concentration of guanosine3′,5′-cyclic monophosphate (cyclic GMP) in each lysate was determinedusing liquid chromatography with tandem mass spectrometry. The data wereused to construct dose-response curves and calculate half-maximaleffective concentration (EC₅₀) values for each test article.

Linaclotide and Cys₁-IMD showed GC-C agonist activity in T84 cells, asmeasured by the increase in intracellular cGMP (FIG. 4). The EC₅₀ valuesfor linaclotide and Cys₁-IMD were 315±105 nM and 172±32 nM,respectively.

Example 4: Measurement of Content and Purity of Exemplary Peptides

Content and purity of the peptides of the present invention may bedetermined by reverse phase gradient liquid chromatography using anAgilent Series 1100 LC System with Chemstation Rev A.09.03 software orequivalent. A YMC Pro™ C18 column (dimensions: 3.0×150 mm, 3.5 um, 120Å; Waters Corp., Milford, Mass.) or equivalent is used and is maintainedat 40° C. Mobile phase A (MPA) consists of water with 0.1%trifluoroacetic acid while mobile phase B (MPB) consists of 95%acetonitrile:5% water with 0.1% trifluoroacetic acid. Elution of thepeptides is accomplished with a gradient from 0% to 47% MPB in 28minutes followed by a ramp to 100% MPB in 4 minutes with a 5 minute holdat 100% M wash the column. Re-equilibration of the column is performedby returning to 0% MPB in 1 minute followed by a 10 minute hold at 100%MPA. The flow rate is 0.6 mL/min and detection is accomplished by UV at220 nm.

Samples for analysis are prepared by addition of the contents ofcapsules of exemplary peptides to 0.1 N HC1 to obtain a targetconcentration of 20 μg peptide/mL. 100 μL of this solution is injectedonto the column.

Cys₁-IMD Peptide

The Cys₁-IMD peptide was purified using a 2-inch Waters C18 column with0.1% TFA buffer with a linear gradient of 15-45% in 60 minutes of bufferB at flow rate of 100 mL/min. The pooled fractions with purity around95% were loaded onto C18 column. After equilibrating the column withTEAP buffer and AA buffer, the peptide was purified and eluted out withHAC buffer with a linear gradient of 15-75% of buffer B in 60 minutes.Pooled fractions with purified peptide were lyophilized to dryness. Anexample of an analysis of linaclotide and Cys₁-IMD product by RP-HPLC isshown in FIG. 2.

Glu3-ethyl Ester Peptide

The Glu3-ethyl ester peptide (6.0 g) was dissolved in 12 L of 0.05Mammonium bicarbonate in water, and the oxidation process was monitoredby Ellman's test, MS and analytical HPLC. The oxidation process tookapproximately 48 hours for completion.

The above solution was filtered and loaded onto a 2-inch C18 column, andpurified by using 0.05M ammonium acetate buffer with a linear gradientof 10-40% of buffer B in 60 minutes at flow rate of 100 mL/min.

The pooled fractions with purity of >95% were lyophilized to dryness.After the peptides were dried, the peptide was re-dissolved inacetonitrile-water and acidified to pH around 4-5 by addition of aceticacid and re-lyophilized to dryness.

Tyr₁₄-ethyl Ester Peptide

The Tyr₁₄-ethyl ester peptide was purified by dissolving 2.5 g of theisolated peptide in 5 L of 0.05M ammonium bicarbonate in water, and theoxidation process was monitored by Ellman's test, MS and analyticalHPLC. This oxidation process took approximately 16 hours for completion.

The above solution was filtered and loaded onto a 2-inch Polymer column,and purified by using 0.05M ammonium bicarbonate buffer with a lineargradient of 15-45% of buffer B in 60 minutes at flow rate of 100 mL/min.The pooled fractions with the peptide were lyophilized to dryness. Afterthe peptide was dried, the peptide was re-dissolved inacetonitrile-water and acidified to pH 4-5 by addition of acetic acidand re-lyophilized to dryness.

The contents of the purified peptides were measured by determining thepeptide concentration in the prepared sample against a similarlyprepared external peptide standard.

Example 5

The effect of including various formaldehyde scavenger compounds on thestability of the linaclotide composition was next assessed.

60 cc square HPDE bottle (with 33 mm caps with induction seals) wereloaded with (i) capsules containing beads coated with 145 μg or 290 μgof linaclotide, a binder, calcium chloride and leucine with anapproximate molar ratio of CaCl₂: leucine: linaclotide of 60:30:1, (ii)either no formaldehyde scavenger compound (negative control) or acanister comprising one of the formaldehyde scavenger compoundsdisclosed in Table 3, and (iii) optionally a canister containing 3 gramsof a silica gel desiccant.

In addition, some of the linaclotide compositions contained aformaldehyde stressor (in particular, an adhesive label on the desiccantcanister that was applied by the manufacturer of the desiccant canister(Sud Chemie) (its presence in some linaclotide compositions is indicatedin the table as “labeled canister”). Regarding the formaldehyde stressorcanister, it was observed by applicants that the canister emits about27.5 ug of formaldehyde in 24 hours at 60° C.

The linaclotide compositions were stored in a 40° C., 75% relativehumidity stability chamber. Next, the quantity of Cys₁-IMD in eachlinaclotide composition was determined following 4, 8 and 13 weekstorage via Reverse Phase-HPLC using a gradient elution with UVdetection at 220 nm. Quantitation was based on percent of chromatogramarea.

Test samples of linaclotide capsules are prepared by emptying the beadcontents into flasks, adding diluent and shaking for 30 minutes. Theanalysis used the bead contents of a composite of 6 or 8 capsules (for290 μg and 145 μg, respectively). Operating conditions for theanalytical test method by HPLC are outlined in Table 2. The results ofthe stability assay are set forth in Table 3.

TABLE 2 HPLC Purity and Degradation Conditions Mobile Phase A 98:2:0.1Water:Acetonitrile:FA Mobile Phase B 95:5:0.1 Acetonitrile:Water:TFADiluent 0.1N Hydrochloric Acid Gradient Time Profile (min) % A % BComments 0 100 0 Initial Conditions 4 100 0 4-minute hold 9 90 10 5-minute linear gradient 43 77 23 34-minute linear gradient 49 66 34 6-minute linear gradient 59 20 80 10-minute linear gradient 60 100 0Return to initial conditions 67 100 0 Re-equilibration UV Detection 220nm Injection 50 uL Volume Sample 200 ug/mL Concentration Column YMC ProC18, 150 mm × 3.0 mm ID, 3 um or equivalent Column 40° C. TemperatureFlow Rate 0.6 mL/min

TABLE 3 Effect of formaldehyde scavenger compounds on the LinaclotideComposition Stability % decrease in Cys₁-IMD as compared % Cys₁-IMDafter to negative storage control Formaldehyde Scavenger 4 8 13 (afterCompound Weeks Weeks Weeks 13 weeks) negative control 1.21 1.87 3.53 —(labeled canister with no scavenger) d-meglumine 0.59 0.81 0.84 −76%(coated on beads in labeled canister) histidine 0.60 0.68 1.00 −72%(coated on beads in labeled canister) lyophilized leucine 0.52 0.78 1.36−61% (in labeled canister) asparagine 0.56 1.11 1.45 −59% (coated onbeads in labeled canister) glycine-leucine 0.61 1.03 1.51 −57% (coatedon beads in labeled canister) lysine 0.87 1.27 1.87 −47% (coated onbeads in labeled canister) glycine-glycine 0.89 1.40 1.98 −44% (coatedon beads in labeled canister) leucine 0.84 1.26 2.44 −31% (coated onbeads in labeled canister) aspartame 0.82 1.54 2.58 −27% (coated onbeads in labeled canister)

As is demonstrated in Table 3, linaclotide compositions that containedformaldehyde scavenger compounds exhibited surprisingly and unexpectedlyhigher stability that those not containing a formaldehyde scavengercompound (negative control).

All publications and patents referred to in this disclosure areincorporated herein by reference to the same extent as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference. Should themeaning of the terms in any of the patents or publications incorporatedby reference conflict with the meaning of the terms used in thisdisclosure, the meaning of the terms in this disclosure are intended tobe controlling. Furthermore, the foregoing discussion discloses anddescribes merely exemplary embodiments of the present invention. Oneskilled in the art will readily recognize from such discussion and fromthe accompanying drawings and claims, that various changes,modifications and variations can be made therein without departing fromthe spirit and scope of the invention as defined in the followingclaims.

What is claimed is:
 1. A linaclotide composition comprising linaclotide,a sterically hindered primary amine, a divalent metal cation and aformaldehyde scavenger compound.
 2. The composition of claim 1, whereinthe composition further comprises a peptide having the structure offormula (I) or a pharmaceutically acceptable salt thereof:


3. The composition of claim 2, wherein the linaclotide compositioncomprises the peptide of formula (I) in a concentration of up to 5% byweight as compared to the weight of linaclotide.
 4. The composition ofclaim 2, wherein the linaclotide composition comprises the peptide offormula (I) in a concentration of up to 3% by weight as compared to theweight of linaclotide.
 5. The composition of claim 2, wherein thelinaclotide composition comprises the peptide of formula (I) in aconcentration of up to 2% by weight as compared to the weight oflinaclotide.
 6. The composition of claim 2, wherein the linaclotidecomposition comprises the peptide of formula (I) in a concentration ofup to 1% by weight as compared to the weight of linaclotide.
 7. Alinaclotide composition comprising a formaldehyde scavenger compound andan oral pharmaceutical dosage form comprising about 145 μg or about 290μg of linaclotide or a pharmaceutically acceptable salt thereof, Ca²⁺ ora salt thereof, leucine, and a peptide having the structure of formula(I) or a pharmaceutically acceptable salt thereof:


8. The composition of claim 7, wherein the peptide of formula (I) ispresent in the oral pharmaceutical dosage form in an amount of about0.001 to about 2 wt. % relative to the total weight of the dosage form.9. The composition of claim 7, wherein the peptide of formula (I) ispresent in the oral pharmaceutical dosage form in an amount of about0.001 to about 1 wt. % relative to the total weight of the dosage form.10. The composition of claim 7, wherein the peptide of formula (I) ispresent in the oral pharmaceutical dosage form in an amount of about0.001 to about 0.75 wt. % relative to the total weight of the dosageform.
 11. The composition of claim 7, wherein the peptide of formula (I)is present in the oral pharmaceutical dosage form in an amount of about0.001 to about 0.5 wt. % relative to the total weight of the dosageform.
 12. The composition of claim 2, wherein the divalent metal cationis Ca²⁺ or a salt thereof, the sterically hindered primary amine isleucine, and wherein the linaclotide composition comprises a molar ratioof divalent metal cation:sterically hindered primary amine:linaclotideof about 57-63:28-32:1
 13. The composition of claim 7, wherein the Ca²⁺or a salt thereof and leucine are present in the oral pharmaceuticaldosage form in a molar ratio of Ca²⁺:leucine:linaclotide of about57-63:28-32:1.
 14. The composition of claim 9, wherein the Ca²⁺ or asalt thereof and leucine are present in the oral pharmaceutical dosageform in a molar ratio of Ca²⁺:leucine:linaclotide of about57-63:28-32:1.
 15. The composition of claim 10, wherein the Ca²⁺ or asalt thereof and leucine are present in the oral pharmaceutical dosageform in a molar ratio of Ca²⁺:leucine:linaclotide of about57-63:28-32:1.
 16. The composition of claim 11, wherein the Ca²⁺ or asalt thereof and leucine are present in the oral pharmaceutical dosageform in a molar ratio of Ca²⁺:leucine:linaclotide of about57-63:28-32:1.
 17. The composition of claim 9, wherein the oralpharmaceutical dosage form further comprises an oxidation product havinga structure of:

in an amount from about 0.05% to about 5% by weight relative to thetotal weight of the dosage form.
 18. The composition of claim 17,wherein the oxidation product in present in the dosage form in an amountof about 0.05% to about 2% relative to the total weight of the dosageform.
 19. The composition of claim 17, wherein the oxidation product inpresent in the dosage form in an amount of about 0.05% to about 1%relative to the total weight of the dosage form.
 20. The composition ofclaim 7, wherein the Ca²⁺ or a salt thereof and leucine are present inthe oral pharmaceutical dosage form in a molar ratio of Ca²⁺:leucine:linaclotide of about 57-63:28-32:1, wherein the formaldehydescavenger compound is selected from the group consisting of d-meglumineand histidine, and wherein the peptide of formula (I) is present in theoral pharmaceutical dosage form in an amount of about 0.001 to about 1wt. % relative to the total weight of the dosage form.
 21. Thecomposition of claim 20, wherein the peptide of formula (I) is presentin the oral pharmaceutical dosage form in an amount of about 0.001 toabout 0.75 wt. % relative to the total weight of the dosage form. 22.The composition of claim 20, wherein the peptide of formula (I) ispresent in the oral pharmaceutical dosage form in an amount of about0.001 to about 0.5 wt. % relative to the total weight of the dosageform.
 23. The composition of claim 1, wherein the formaldehyde scavengercompound is selected from the group consisting of leucine, glycine,aspartame, d-meglumine, histidine, asparagine, proline, or a mixturethereof.
 24. The composition of claim 23, wherein the formaldehydescavenger compound is d-meglumine, histidine or a mixture thereof. 25.The composition of claim 23, wherein the formaldehyde scavenger compoundis d-meglumine.
 26. The composition of claim 23, wherein theformaldehyde scavenger compound is histidine.
 27. The composition ofclaim 28, wherein the formaldehyde scavenger compound is present in thelinaclotide composition in molar ratio of formaldehyde scavengercompound to linaclotide between 3:1 and 1:1.